Transformation of plants to introduce closely linked markers

ABSTRACT

A novel method of producing a plant with a marker closely linked to a target locus, in particular a nuclear male sterile target locus, is described. The method involves transformation of a group of plants in order to introduce a marker into each plant, and isolation of a plant with the marker closely linked to a target locus. The markers include visible markers and dominant conditional lethal markers. The method is of particular use for hybrid seed production where the target locus is a nuclear male sterile locus.

FIELD OF INVENTION

This invention relates to a method of producing a plant with a markerclosely linked to a target locus, in particular a nuclear male steriletarget locus. This invention more particularly relates to transformationof a group of plants in order to introduce a marker into each plant, andisolation of a plant with the marker closely linked to the target locus.The invention is of particular use for hybrid seed production where thetarget locus is a nuclear male sterile locus.

BACKGROUND OF THE INVENTION

The use of hybrid plants in agricultural crops is well known as a meansto enhance plant production and value. A hybrid plant is one resultingfrom a crossing (an outcrossing) other than a self-crossing (selfing) ora sibling-crossing (sibbing), in particular between inbred lines. Hybridcrossing, or hybridization, is commonly accomplished by having side byside stands or rows ("crossing" rows), with female stands (P1)alternating with pollinating rows (P2). Pollen is normally carried byinsects or wind, although in some commercial plants pollination is byhand.

The value of the hybrid is primarily in the increased yield and vigor,or heterosis, which is displayed as compared to the parents, inparticular in the first generation (F1) of progeny. Selfing of a hybrid(F1) results in a second generation of plants (F2) which normallyexhibits less heterosis than is displayed by the F1 generation. Hybridshave been commercially used in a great variety of plants and crops,including wheat, field corn, sweet corn, barley, sorghum, sugar beets,garden beets, onions, tomatoes, cabbage, cauliflower, broccoli, brusselssprouts, cucumber, carrot, spinach, summer and winter squash, asparagus,pepper, eggplant, radish, muskmelon, watermelon, pumpkin, cantaloupe,tobacco and various ornamentals.

In developing hybrid seed (i.e., seed from which the first generationhybrid is to be grown), the ability to control mass pollination isimportant. Specifically, prevention of parental selfing or sibbing,which would result in the production of non-hybrid seed, is important.Several methods have been used, or considered, for ensuring ormaximizing hybridization.

One method is to emasculate the female parent line (P1), either by handor mechanically. For instance, in the alternating stand situation, ifthe P1 stand is emasculated, plants in that stand can only be pollinatedby plants from another stand, i.e., the pollinating stand. While thishas been done with some plants, e.g., tomato (manual) and corn (manualand mechanical), manual emasculation is labor intensive and impracticalfor volume crops, and mechanical emasculation cannot be used for manycrops and can cause yields to suffer due to plant damage.

A second method is the use of chemical agents (gametocides) toemasculate the intended female parents. Although attempts have been madeto develop such chemicals, the approach has a number of problems. Thechemical must provide sufficient male sterility without otherwiseaffecting the plant. There is the need to treat selectively only theultimate female parents and to spray only in the proper amounts. Plantssprayed with insufficient chemical or sprayed at the wrong stage ofdevelopment may not be rendered male sterile (so-called "shedders"). Inaddition, the cost of the gametocide may be a factor.

A third method applies to those plants in which cytoplasmic malesterility (cms) has been found, e.g., cotton, tobacco, rice, corn,onions, sorghum, carrot, radish, alfalfa and certain flowers andgrasses. For such plants, cms in the female parent will ensure itsoutcrossing. The approach is limited in that, in general, the occurrenceof cms in crops is rare. Also, for those crops in which the productdepends on fertilization the use of cms is workable only if a restorergene is available for the particular plant to permit restoration offertility of the F1 hybrid. In addition, cms is often associated withundesirable traits, some of which may not appear at the outset. Forinstance, a cms system used in maize led to the Southern leaf blightepidemic of the early 1970's. U.S. Pat. No. 2,753,663 discloses the useof cms in corn.

A fourth method which has been considered is the use of nuclear malesterility, also referred to as chromosomal or genic male sterility.Nuclear male sterile genes presumably result from a mutation at somepoint in the development of a plant or its ancestors. The genes occur inpractically all diploid plant species which have been examined for thisproperty, and are present as well in polyploid plants. The genes arenormally recessive. Nuclear male sterile genes have been studied in alarge number of plants of agricultural interest, e.g., wheat, corn,tomato, barley, pepper, rice, lima beans, peas, cotton, watermelon,soybeans, tobacco, lettuce and various ornamentals.

See R. Frankel et al., Pollination Mechanisms, Reproduction and PlantBreeding, (Springer Verlag), 3.4.1.2 and 3.4.2.1 (1977) See W.Gottschalk et al., "Induced Mutations in Plant Breeding", Monographs onTheoretical and Applied Genetics, 7, 70 (Table 13) (1983) for a list ofinduced male sterile mutants in cereals and dicotyledonous plants. Formany plants there are a number of male sterile loci, each of which canhave either a recessive male sterile (ms) or a dominant male fertile(Ms) allele. For a diploid plant, such a locus (allele pair) can behomozygous male sterile (ms/ms), homozygous male fertile (Ms/Ms), orheterozygous male fertile (Ms/ms). The nomenclature shown is forrecessive male sterility. In the less likely instance of dominant malesterility, the nomenclature would be the opposite (Ms male sterile; msmale fertile). Unless otherwise stated, the nomenclature used herein isfor recessive male sterility. Nuclear male sterility and nuclear malesterile plant, line, gene, locus, etc. shall on occasion be referred toherein, respectively, as male sterility and male sterile plant, line,gene, locus, etc.

Nuclear male sterility has use in hybrid breeding in that if the femaleparent displays nuclear male sterility (e.g., in a diploid plant, if theplant is homozygous for recessive ms), the female parent cannot functionas a male parent and seeds obtained from it by outcrossing arenecessarily hybrid seeds. In the alternating stand situation, nuclearmale sterile female stands alternate with pollinating stands. Seedharvested from the female stands can be used as hybrid seed.

The use of nuclear male sterility in hybrid breeding requires a means tomaintain the nuclear male sterile line. Since male steriles produce nopollen, to be maintained they must be produced anew each generation fromsegregating progenies. For a diploid plant, a maintainer line istypically obtained by crossing the male sterile line (ms/ms) with a malefertile line (Ms/Ms). The product is a heterozygous maintainer line(Ms/ms) which is used to generate the next generation male sterile linein a crossing to a homozygous male sterile line (ms/ms). The progeny ofthis crossing are 50% homozygous male sterile (ms/ms) and 50%heterozygotes (Ms/ms). In such a situation there must be a means toisolate the male sterile plants for use as the female parent in hybridbreeding. This has to a large extent been done by visual inspection,although there have been suggestions for using either natural linkage orchromosomal abnormalities to assist in isolating male steriles. See, ingeneral, J. R. Welsh, Fundamentals of Plant Genetics and Breeding,Chaps. 16, 18 John Wiley and Sons, (1981) for a discussion of hybridbreeding and male sterility systems.

The visual inspection approach to isolating male steriles involvesexamining individual plants in the field at the time of anthesis. Malefertiles often show physical differences, e.g., in anther structure,which permit them to be mechanically or manually removed (rogued). Whilethis approach has been used for some crops, it is not effective for manyplants of agronomic interest. Moreover, it is labor intensive and cannotbe used for selection at an early stage of plant development.

Another approach to obtaining male sterile stands involves the use ofnaturally occurring linkage. As used herein, the term linkage meansgenetic linkage between two or more loci (or genes) in a given genomesuch that the recombination frequency between the loci (or genes) isless than 50%, the frequency of random assortment. As so used, the termusually refers to loci (or genes) in sufficiently close proximity on thesame chromosome of a given plant that recombination between them occursless frequently than if the genes were located on separate chromosomes.The term, as used herein, also embraces other forms of genetic linkagewhere the recombination frequency deviates from random assortment. Theextent of linkage is measured in terms of the recombination frequency,or recombination units. Linked genes are normally transmitted orinherited together at a frequency related to their recombinationfrequency, as can be shown by segregation analysis of progeny resultingfrom a crossing. If a chromosomal recombination (a crossover or otherrearrangement event) takes place, linked alleles may not be transmittedtogether. The tighter the linkage, the less likely is a recombinationresulting in disruption of the linkage. For instance, very tight linkageexists if the linked genes are adjacent to each other on the samechromosome. As stated, the extent of linkage is measured in terms ofrecombination units. If two genes are separated by ten recombinationunits, there is a 10% chance of their becoming uncoupled in a crossing.A separation of five recombination units constitutes a tighter linkage.

There have been a number of proposals for using naturally occurringlinkages between a male sterile gene and a marker gene (i.e., linkages,involving male sterile mutants, either discovered in nature or resultingfrom a breeding program) as a means of detecting or isolating malesterile plants or seeds from a mixture of male steriles and malefertiles. There is a 1930 disclosure of the possibility of usingnaturally occurring linkage between nuclear male sterility and seedcolor in bicolor corn in a program to produce hybrid corn seed; W. R.Singleton et al., Journal of Heredity, 21, 266-68 (1930). Otherdisclosures concern the use of natural linkage in barley between malesterility and recessive DDT resistance as a means of removing malefertiles (upon application of DDT) in a hybrid program; G. A. Wiebe,Argon. J., 52, 181-82 (1960) and G. A. Weibe, Barley Newsletter, 8, 16(1964). Wiebe (1964) also discloses the possibility of reducing thecrossover value (that is, tightening the linkage) either by usingradiation to invert chromosomal segments or by breeding fortranslocations. Use of linkage between a male sterility gene and a genefor non-germination upon treatment of a particular chemical is suggestedin R. T. Ramage, Barley Newsletter, 9, 3-8 (1966). Linkage between malesterility and color, or lack-of-color, genes is disclosed in J.Philouze, Ann. Amel. Plant., 24, 77-82 (1974), cited in M. Yardanov,Monographs on Theoretical and Applied Genetics, 6, 189-219 (1983). Suchlinkage is also disclosed in C. A. Foster, Barley Genetics Newsletter,9, 22-23 (1979). Linkage between a male sterile locus and a shrunkenseed gene is disclosed in D. E. Falk et al., Barley Genetics, IV, 778-85(1981). A tomato mutant with linkage between an isozyme marker and amale sterile locus resulting from a breeding program is disclosed in S.D. Tanksley, Plant Molecular Biology Reporter, 1, 3-8 (1983) and C. M.Rick et al., Isozymes: Current Topics in Biological and MedicalResearch, 1, 269-84 (1983). Natural linkage is also disclosed in C. A.Foster, Barley Genetics, III, 774-84 (1976); and R. T. Ramage,Monographs on Theoretical and Applied Genetics, 6, 71-93 (1983). Themarker is termed a "haplo-viable" mutation (Ramage, at 84); it is linkedto the dominant allele of the male sterile locus. So-called haplo-viablemutations can be transmitted through the egg but not through the pollen.The use of natural linkage to assist in isolating male steriles islimited by the lack of appropriate markers for many crops ofagricultural interest and also, for those crops for which markers areknown, by the lack of sufficient closeness of linkage.

Another approach for obtaining male sterile lines which has beensuggested involves the use of chromosomal abnormalities. One type ofabnormality is a differentially transmitted chromosome, e.g., aduplicate deficient chromosome (egg-viable but not transmitted bypollen) as disclosed in U.S. Pat. No. 3,710,511. This chromosomalvariation is disclosed as occurring naturally or resulting frommutagenic agents. If a female stand is produced from a Ms/msheterozygote in which the male fertile allele is linked with adifferentially transmitted chromosome (transmitted through the femaleparent but not the male parent), selfing or sibbing can be avoided.Another type of chromosomal abnormality is the presence of an extrachromosome, meaning the presence of an additional chromosome from thesame species or the presence of an additional chromosome from a relatedspecies. R. T. Ramage, Barley Newsletter, 9, 3-8 (1966) discloses asystem in barley with an extra chromosome (a trisomic system) resultingfrom disjunction during mitosis. The extra chromosome is transmittedthrough the female germ line but only rarely transmitted through themale. Fertility genes and marker genes (e.g., phytocide susceptibility,height, seed size or shape) on the extra chromosome permit productionand identification of male sterile female parents. C. J. Driscoll, CropSci., 12, 516-17 (1972) discloses an analogous system for hybrid wheatusing an extra chromosome derived from rye. U.S. Pat. No. 4,051,629 alsodiscloses an extra chromosome system. In addition, systems with extrachromosomes are disclosed in R. Frankel et al., Pollination Mechanisms,Reproduction, and Plant Breeding, (Springer Verlag), §3.4.4 (1977); P.Wilson et al., "Hybrid Wheat", Monographs on Theoretical and AppliedGenetics, Chap. 4, 94-123 (1983); and J. Sybenga, Theor. Appl. Genet.,66, 179-201 (1983). Sybenga, at 194, suggests the further development ofan extra ("alien") chromosome system by constructing such a chromosomeusing molecular genetic engineering. In general, the extra chromosomeapproach is limited (a) by difficulties in deriving the appropriatesystem, as well as (b) by problems of low pollen yield.

SUMMARY OF THE INVENTION

In accordance with the invention, a method is provided for producing anovel plant with a marker gene (marker) closely linked to a targetlocus, in particular where the target locus is a male sterile locus. Amarker closely linked in this manner shall be referred to herein onoccasion as a close-linkage marker and a plant so produced, or progenyof such a plant, shall be referred to as a close-linkage plant. Thenovel plant is obtained by transforming a first plant with a markerusing genetic engineering techniques to produce a group of transformedplants (transformants) containing the marker at various positions withinthe plant genome of different transformants. Selection is made fortransformants containing the introduced marker at a single locus withinthe transformant (i.e., single locus transformants). The selection forsingle locus transformants is preferably accomplished by conductingcrossings, either self-crossings or outcrossings (for instance,outcrossing with homozygous male sterile plants), of each transformant,and scoring (analyzing or testing) the progeny of the crossings forpresence of marker to determine, based on segregation patterns, whethera given transformant is a single locus transformant. Multiple locustransformants (transformants other than single locus transformants) areeither discarded or converted to single locus transformants by furthercrossing and progeny testing. From the group of single locustransformants, selection is made for those plants containing the markerin close or tight linkage with the target locus. Where the target locusis a male sterile locus, the marker can be linked either to the malesterile allele or the male fertile allele, as long as it is linkage incoupling phase (dominant alleles of marker and male sterile loci on samechromosome). If the linkage is in repulsion rather than in couplingphase, the phase can be reversed by a separate recombination crossingstep.

The selection for close-linkage plants is made by conducting crossingsof each single locus transformant (e.g., for a male sterile targetlocus, crossings with homozygous male sterile plants followed by selfingor backcrossing of the hybrid), and by scoring the progeny to determine,based on the segregation pattern, whether the marker is closely linkedto the target locus. The greater the association between the marker andthe target locus in the segregation distribution, the closer thelinkage.

Where the target locus is a male sterile locus, a male sterile gene canbe present at the time of transformation or it can be subsequentlyintroduced by crossing of the transformant and selection (from the firstor second generation of progeny) of a plant containing the male sterilegene in the desired form (e.g., heterozygous). The first plant (theplant to be transformed) can be homozygous male sterile, homozygous malefertile, or heterozygous.

Transformation of a plant can be carried out using various methods. Apreferred method is the use of the natural vector system Agrobacterium(including both Agrobacterium tumefaciens and Agrobacterium rhizogenes),for instance by cocultivation or inoculation techniques. An especiallypreferred method is transformation using Agrobacterium tumefacienscontaining Ti plasmid vectors, where the vectors contain the markerwithin the T region but the tumor-causing genes are removed orinactivated (i.e., the vector is disarmed). Transformation usingtransposons can also be used. In addition, transformation may be bytake-up of naked DNA. Transformed plants can be regenerated using knowntechniques. The method of the invention can be used with plants amenableto transformation and regeneration techniques. The invention embracesnovel close-linkage plants, or seeds, produced by this method.

Markers for use with the invention can be either expressive markers orconditional expressive markers. Expressive markers include visiblemarkers and assayable markers. Conditional expressive markers includeconditional color markers (chromogenic markers), conditional resistancemarkers (e.g., antibiotic resistance or herbicide resistance), anddominant conditional lethal markers. When closely linked to a malesterile locus, these markers permit detection of male sterility, eitherdirectly or conditionally upon application of a chemical, preferably atan early stage of plant development, i.e., at a stage before anthesis,and most preferably at the seed or seedling stage. The inventionembraces plants transformed with these markers.

In accordance with the invention, close linkage between marker and atarget locus can be generated at various levels of closeness, dependingon the number of transformants and the number of crossings involved inselecting a close-linkage plant. Depending on the intended use of theclose-linkage plant, linkages of about 20 units can be useful. Ingeneral, and particularly where the target locus is a male sterilelocus, the linkage is preferably less than about 10 units, with linkagesof less than about 5, 3, or 1 unit, respectively, being increasinglypreferred.

In accordance with the invention the means is provided to create closelinkage between a marker gene and a target locus in a wide variety ofplants, including those for which such linkage was not previously knownin nature. The invention is of particular use for plants which reproduceby seed (i.e., sexually reproducible plants). The invention provides themeans to create linkage at levels of closeness of use as a generalbreeding tool, e.g., to survey for male sterile parents. In particular,the invention is of use in hybrid breeding as a means to isolate andseparate male sterile plants for use as female parents. Through use ofthe invention pure or substantially pure rows of male sterile plants canbe produced for use as female parents in production of substantiallypure hybrid seed.

Where the close-linkage plants are used in hybrid breeding, theclose-linkage plant is preferably used as, or converted to by crossing,a maintainer line heterozygous for male fertility and heterozygous forthe marker. The maintainer line can be crossed with a homozygous malesterile line to yield a 50:50 mixture of male fertile and male sterileplants. The male fertiles are removed from the mixture based upon thepresence of the marker. If the marker is an expressive marker, thefertiles are removed based on presence or absence of color. If themarker is a conditional expressive marker, all the plants in the mixtureare subjected to the applicable chemical and fertiles are removed basedon the appearance, or lack of appearance, of color (if the marker is achromogenic marker); slight plant damage (if the marker is a resistancemarker); or lethality (if the marker is a dominant conditional lethalmarker). With male fertiles removed, the remaining substantially puregroup of male steriles can be used as the female plants in hybridcrossing The female plants are pollinated with pollen from an inbredline and hybrid seed is harvested for sale or planting. The maintainerline can be maintained indefinitely by backcrossing and selection ofmaintainer progeny.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a depiction of steps involved in selection of plants made inaccordance with the invention.

FIG. 2 is a schematic providing restriction information for plasmidspAGS751, pAGS753, pAGS754, pAGS755 and pAGS756.

FIG. 3 is a schematic showing the preparation and restriction patternsof plasmids pAGS755 and pAGS756.

FIG. 4 is a schematic showing restriction information for plasmidspAGS757, pAGS758 and pAGS759.

DETAILED DESCRIPTION

The invention comprises a method of producing novel plants, the novelplants so produced, and methods of using the novel plants. As usedherein, the term plant means either a whole plant, a plant part, a plantcell, or a group of plant cells. The class of plants which can be usedin the method of the invention is as broad as the class of higher plantsamenable to transformation techniques. It includes plants of a varietyof ploidy levels, preferably diploid. Polyploids best lend themselves touse in the invention if they are functional diploids, e.g., for atetraploid, if there are two chromosome pairs with one pair homozygousms/ms and the other pair heterozygous Ms/ms. The class of plants alsoincludes plants which contain one or more target loci or in which targetloci can be introduced. The invention has particular application toplants which benefit, or stand to benefit, from hybrid breedingtechniques.

The invention is further described with particular reference to malesterile loci as the target loci, e.g., for use in hybrid breeding, butwith the understanding that other target loci could be employed. Forinstance, in plant breeding, the locus could be any locus of interest(e.g., disease resistance) which the breeder desires to follow by thepresence of close-linkage markers. Close-linkage plants for use inhybrid breeding are of importance for plants of agricultural interest,including dicotyledenous plants such as tomato, tobacco, cotton,rapeseed, field beans, soybeans, peppers, lettuce, peas, alfalfa,clover, cole crops or Brassica oleracea (e.g., cabbage, broccoli,cauliflower, brussels sprouts), radish, carrot, beets, eggplant,spinach, cucumber, squash, melons, cantaloupe, sunflowers and variousornamentals, and monocotyledenous plants such as asparagus, field orsweet corn, barley, wheat, rice, sorghum, onion, pearl millet, rye andoats.

The invention may be used with plants which have a target locus, e.g., amale sterility locus, which is found to exist in nature, which isintroduced by mutagenesis, which is introduced to the plant throughbreeding, or which is otherwise introduced. In nature, male sterilityoccurs spontaneously in many field plants at a frequency of 1 in 10⁴ to1 in 10⁵, and thus is easy to isolate. Techniques of mutagenesis canyield

frequencies on the order of 1 in 10³. There can be a single male sterilelocus or multiple loci. If there are more than one male sterile loci,the phrase heterozygous for male fertility means heterozygous for atleast one of the loci.

Many plants have been studied genetically in detail and information isavailable about male sterile loci and their positions. In tomato, a wellstudied species, there are over thirty known male sterile genes; in cornthere are approximately fifteen, in barley over twenty, and in sorghumsix. Male sterile genes have also been studied in rice, cotton, pea,soybean, tobacco, pepper, eggplant and lettuce. Male sterile lines ofthese plants are available.

As stated, male sterility is normally recessive, and thus is notexpressed unless the plant is homozygous ms/ms. The method of theinvention has particular application for plants with such recessive malesterile alleles. The method can also be used, however, for dominant malesterile alleles in crops where seed is not an important product, e.g.,spinach, cabbage, lettuce, broccoli, cauliflower and brussels sprouts.

The transformation of plants in accordance with the invention may becarried out in various ways known to those skilled in the art of plantmolecular biology. As used herein, the term transformation meansalteration of the genotype of a host plant by the introduction offoreign DNA such that it is integrated into the genome. The termtransformable plant, as used herein, means a plant which can betransformed. Normally, regeneration will be involved in obtaining awhole plant from the transformation process. The term regeneration, asused herein, means growing a whole plant from a plant cell, a group ofplant cells, a plant part or a plant piece (e.g., from protoplast,callus, or tissue part).

As used herein, the term marker refers to a gene (or group of two ormore genes) conditioning, or encoding, a trait (or a phenotype) whichpermits the selection of, or the screening for, a plant containing themarker. There are two categories of markers of use with the invention:expressive markers and conditional expressive markers.

Expressive markers are markers which are expressed without chemicaltreatment, with the expression product either being directly observable,or assayable. One type of expressive marker is a visible marker, i.e., amarker for a visible trait such as color, fluorescence or plantmorphology. Such a marker can be visible throughout the plant, orlocalized on a particular plant part, e.g., leaf, stem, flower,endosperm, cotyledon or aleurone (seed). A visible marker of choice is acolor marker, preferably one which is identifiable before pollen release(anthesis), e.g., at the seed or seedling stage. A visible marker can beeither dominant or recessive. If the visible marker is dominant, it islinked in coupling with the dominant male fertile allele (where thetarget locus is a male sterile locus); if recessive, it is linked incoupling with the recessive male sterile allele.

An example of an expressive marker which is a visible marker is the genefor naphthalene dioxygenase, an enzyme which converts indole into thepigment indigo. The enzyme is encoded for by bacterial genes found incertain strains of Pseudomonas putida, e.g., PpG7. Indole may be presentin plant tissue or seeds and, if converted to indigo in sufficientamounts, the indigo is detectable in the plant visibly (e.g., by opticalscanning device). In the case of seeds, detection may be either directlyor after slitting the plant. If indole is not present in the plant, theintroduction of the procaryotic gene for tryptophanase or tryptophansynthetase (subunit alpha) into the plant by transformation will permitproduction of indole from tryptophan or indole-3-glycerol phosphate,respectively, already present in the plant. Napthalene dioxygenase isencoded by an operon, i.e., a group of genes (in this case, four), eachencoding a subunit of the enzyme. See B. Ensley et al., Science, 222,167-69 (1983). Depending on the particular plant being transformed, someor all of the genes in the group can be introduced.

Another type of expressive marker is an assayable marker. An example ofsuch a marker is a gene for an enzyme that brings about the synthesis ofan opine (e.g., nopaline) by the plant. The nopaline marker (gene fornopaline synthase) is present in some Agrobacterium Ti plasmids withinthe T region (nos Ti plasmids). See P. Zambryski et al., EMBO J., 2,2143-50 (1983); L. Otten et al., Biochim Biophys. Acta., 527, 497-500(1978). Such a marker can be linked to a male sterile locus inaccordance with the invention.

A conditional expressive marker is a marker, the presence of which isdetectable or assayable in the plant (or in tissues removed from theplant) upon the application of a chemical. That is, the expression ofsuch a marker is conditional upon application of a chemical. As usedherein, the term conditional expressive marker embraces chromogenicmarkers, resistance markers and dominant conditional lethal markers.

A chromogenic conditional expressive marker is one which yields adetectable color upon application of a color-developing agent. Themarker can be dominant or recessive, as long as it is in coupling phasewith the male sterility locus (where the target locus is a male sterilelocus). Such a marker, when closely linked to a male sterility locus,permits identification of male fertiles or male steriles based uponcolor. If it is the male sterile in which color develops, i.e., if themarker is recessive, dosages must be available such that colordevelopment is observable but not substantially deleterious to seed set,seed development or seed production.

A preferred chromogenic marker is beta-lactamase. This enzyme, encodedfor by dominant bacterial genes found in a wide variety of bacteriaincluding E. coli, brings about color change in a number of differentsubstrates. See A. Bourgault et al., J. Clinical Microbiology, 9, 654-56(1979). For instance, nitrocefin is converted from yellow to red, K.Shannon et al., J. Antimicrobial Chemotherapy, 6, 617-21 (1980);pyridene-2-azo-p-dimethylaniline cephalosporin (PADAC) is converted froma purple to yellow, R. N. Jones et al., J. Clinical Microbiology, 15,677-83 (1982); and benzylpenicillin, in the presence of bromcresolpurple, is converted from purple to yellow, W. L. Boyko, Anal. Biochem.,12, 85-88 (1982). A plant transformed with beta-lactamase, uponapplication of any of the above substrates, will be assayable based onthe color change. Close linkage to male fertility/male sterility willpermit a corresponding assay for male steriles.

Another chromogenic marker of choice is the gene for beta-galactosidase.This enzyme, encoded by dominant bacterial genes found in variousbacteria including E. coli, converts o-nitrophenyl-beta-D-galactoside(ONPG) to yellow; 5-bromo-4-chloro-3-indolyl-beta-D-galactoside (XG) toblue; and 6-bromo-2-naphthyl-beta-D-galactoside, in conjunction with4-benzoylamino-2,5-dimethoxyaniline, to blue. See J. H. Miller,Experiments in Molecular Genetics, Cold Spring Harbor Laboratory, NewYork, pp. 48-49, 54-55 (1972).

A resistance conditional expressive marker is a marker which permitsassay for its presence by application of the chemical, e.g., anantibiotic or a herbicide, for which resistance is provided by themarker. The chemical to be applied to the plant is one which in theabsence of resistance confers some observable damage to the plantwithout unduly weakening it. The marker is normally dominant forresistance and is in coupling phase with the male fertility allele towhich it is linked (where the target locus is a male sterile locus). Ifthe conditional expressive resistance marker is recessive, it can alsobe used in accordance with the invention by linkage in coupling phase,which will permit identification and removal of the male fertiles(sensitive). The test for resistance is administered by applying theantibiotic (e.g., kanamycin) to the plant at a dosage independentlydetermined to cause detectable but non-systemic tissue necrosis. Thatis, the effect of the antibiotic on the sensitive plants (e.g., the malesteriles when closely linked) is visible but non-lethal tissuediscoloration or necrosis.

An antibiotic resistance marker of choice is kanamycin resistance. Thegene for resistance encodes for aminoglycoside 3'-o-phosphotransferaseII, an enzyme which confers resistance to certain antibiotics includingkanamycin, neomycin and G418. Plants can be tested for resistance by theability of detached shoots to root in the presence of antibiotic (e.g.,kanamycin sulfate at 10 mg/l) on MS agar, or by the ability of anexcised segment of a leaf to form callus on callus medium supplementedwith antibiotic (e.g., kanamycin sulfate at 100 mg/l). Depending on thesystem, another useful way to score presence of a resistance markerwould be to spray leaves of seedlings. The leaves of sensitive plants(corresponding to homozygous nuclear male steriles), upon treatment withantibiotic in appropriate dosage, develop necrotic lesions; plants withresistance (corresponding to the male fertile phenotype) would not. Intomato, for instance, application of kanamycin over a broadconcentration range of approximately 75 to 200 micrograms/ml permitsidentification of plants containing the marker, with full recovery andnormal flowering of the plants. Seedlings are sprayed and after 7-14days the phenotype is scored. Transformation with the gene for kanamycinresistance is described in R. T. Fraley et al., Proc. Natl. Acad. Sci.,80, 4803-07 (1983), and in M. De Block et al., EMBO J., 3, 1681-89(1984).

Another example of a resistance marker is the gene for resistance to theantibiotic chloramphenicol. The gene for resistance, found in entericbacteria including E. coli, encodes chloramphenicol acetyl transferase.See M. De Block et al., EMBO J., 3, 1681-89 (1984); Herrera-Estrella etal., Nature, 303, 209-13 (1983). Other examples are the markers formethotrexate resistance and hygromycin resistance.

Another type of conditional expressive marker is a dominant conditionallethal marker. Such markers are genes encoding enzymes which can converta precursor into a phytotoxin. Upon application of the precursor to theplant, e.g., in a chemical spray, the presence of the marker results inconversion of the precursor into a chemical lethal to the plant. For usewith a male sterile target locus the conditional lethal marker gene isdominant and is linked in coupling to the dominant male fertile allele(a recessive conditional lethal would kill the male steriles). Uponapplication of the precursor, male fertile plants are subjected to theeffects of the toxin whereas male sterile plants are unaffected. Thisprovides a means of obtaining a row of male steriles without need forindividual inspection or physical rogueing.

A broad class of genes, many of them of bacterial origin, can beemployed in a conditional lethal system. In general, the genes for anyenzyme or series of enzymes that function as an antibiotic synthetasecan be conditional lethal markers if the precursor (i.e., the compoundconverted to the antibiotic by the antibiotic synthetase) isnon-phytotoxic and if it is not produced by the plant. The inventionembraces novel plants transformed with dominant conditional lethalmarkers, preferably where the marker is closely linked to a targetlocus.

A conditional lethal marker of choice is the iamH gene, encoding theenzyme indole acetamide hydrolase (IAMH), found in Agrobacteriumtumefaciens. The enzyme can convert acetamides to acetic acids. The geneis available and can also be obtained from Agrobacterium. See D. Inze etal., Mol. Gen. Genet., 194, 265-74 (1984). For instance, (iamH) convertsindole acetamide (IAM) to indole acetic acid (IAA). It also convertsnaphthalene acetamide (NAM) to naphthalene acetic acid (NAA), a compoundof substantially greater toxicity to plants. Tobacco and tomato aresensitive to NAA at a spray dosage above 0.5 mg/ml. If the gene for IAMHis introduced into a plant, e.g., using Agrobacterium, spraying of theplant with NAM will result in the production of NAA and consequent plantdeath. If the plant has the dominant iamH gene linked in coupling phaseto the male fertility allele in accordance with the method of theinvention, spraying a mixture of male fertiles and male steriles withNAM will result in destruction of male fertiles. Other examples ofdominant conditional lethal markers are the genes for methoxininedehydrogenase (converts methoxinine, or 2-amino-4 -methoxy-butanoicacid, to methoxyvinyl glycine in Pseudomonas aeruginosa), R. Margraff etal., Experientia, 36, 486 (1980); and rhizobitoxine synthetase (producesrhizobitoxine, or 2-amino-4-(2-amino-3-hydroxypropoxy)-trans-3-butenoicacid in Rhizobium japonicum), L. D. Owens et al., Weed Science, 21,63-66 (1973).

Dominant conditional lethal markers introduced into plants in accordancewith the invention may serve as selective plant removal agents incontexts other than hybrid breeding. When a target locus other than malesterile is tightly linked in coupling to such a marker, plantscontaining the target locus can be selected (or identified) based on thepresence of the marker in a way analogous to that described herein for amale sterile target locus. In other contexts, dominant conditionallethal markers have uses apart from close linkage. For instance, aground cover crop (or a ground cover which is a common weed) containingsuch a conditional lethal gene, introduced by transformation, can bekilled by application of the appropriate precursor, thereby freeing anentire field for replanting. Another use is to remove volunteerseedlings from the previous crop in a crop rotation situation. Anadditional use is to weaken or remove one crop selectively in apolyculture situation.

Transformation of a plant to insert a marker can be accomplished at thewhole plant level, the organ or tissue level, the cell level or theprotoplast level, using any one of a number of transformation techniquesknown in the art which will lead to integration of the introduced geneinto the nuclear genome of the plant. For instance, as described furtherbelow, Agrobacterium tumefaciens or Agrobacterium rhizogenes can be usedto transform a plant. Transposons can also be used to introduce genesinto plants. Alternatively, naked DNA transformation of plants, e.g.,transformation of plant protoplasts, may be brought about with varioustechniques, for instance, the use of polycations or polyethylene glycol,the use of electroporation (electric fields), or the use of mechanicaldelivery by microinjection. See M. Saul et al., Programme and AbstractsNATO Advanced Studies Institute, FEBS Advanced Course, E15 (1984).

Agrobacterium tumefaciens and Agrobacterium rhizogenes are species ofbacteria which in nature can infect a plant at a wound site, in theprocess causing tumor formation but also introducing some of the DNA ofthe Agrobacterium into the plant for chromosomal take-up. The introducedDNA (transferred DNA, or T-DNA) is carried on the Ti (tumor-inducing)plasmid of Agrobacterium. This system, in particular Agrobacteriumtumefaciens, has been studied and modified for use as a vector system inplant transformation. Techniques are known for deleting that part of theAgrobacterium genome (the onc region) which causes tumor formation(i.e., disarming the plasmid) and for inserting foreign DNA into the Tregion of the Ti plasmid for introduction into the plant. As usedherein, the term T-DNA refers to any DNA carried between and includingthe left border (LB) and right border (RB) sequences of the Ti plasmid.LB and RB are the only sequences required in cis for transfer of DNAfrom Agrobacterium to a plant cell. Other functions, e.g., virulence orvir genes, may be present in trans. In certain Agrobacterium strainscarrying octopine Ti plasmids there are two T-DNA regions, known asTL-DNA and TR-DNA, each bounded by its own LB and RB sequences. InpTi15955, TL and TR are separated by shorter DNA segment called TC-DNA(R. F. Barker et al., Plant Molecular Biology, 2, 335-50 (1983)).

Various plasmid vectors have also been developed for use in the Tisystem. See L. Ream et al., Science, 218, 854-59 (1982); M. W. Bevan etal., Ann. Rev. Genet., 16, 357-84 (1982); P. Zambryski et al., EuropeanPatent Application No. 116,718, published Aug. 29, 1984; R. Fraley etal, Proc. Natl. Acad. Sci., 80, 4803-07 (1983); M. Bevan et al., Nature,304, 184-87 (1983); and P. Zambryski et al., Genetic EngineeringPrinciples and Methods, Vol. 6, J. Setlow et al. eds., 253-78 (1984);Also, see A. Hoekema et al, Nature, 303, 179-80 (1983) regarding use ofa binary vector system in Agrobacterium.

Agrobacterium can be used to transform plants in various ways includinginoculation of wounded plant tissue (e.g., stem inoculation or leaf discinoculation) and protoplast cocultivation. The transformed plants may bedicotyledenous or monocotyledonous (see G. M. S. Hooykaas et al.,Nature, 311, 763-64, (1984). Agrobacterium may be used for the method ofthe invention with any plant which is transformable by Agrobacteriumwith stable uptake into the plant genome and which, where necessary, isregenerable after such transformation.

There have been reports of the use of Agrobacterium to introduce variousgenes into plants. For instance, Agrobacterium has been used tointroduce the chloramphenicol acetyltransferase and octopine synthasegenes in tobacco, with stem inoculation, L. Herrera-Estrella et al.,Nature, 303, 209-13 (1983); the neomycin phosphotransferase II(kanamycin resistance) gene in tobacco with stem inoculation, see M. W.Bevan et al., Nature, 304, 184-87 (1983) and references cited above indiscussion of kanamycin resistance as a marker; and the dihydrofolatereductase gene in tobacco with cocultivation, L. Herrera-Estrella, EMBOJ., 2, 987-95 (1983).

Transformation of plants using Agrobacterium can be carried out usingknown methods of gene enhancement or control. Promoter systems known inthe art can be employed to bring about or ensure adequate expression ofthe marker. Promoters of choice include the nopaline synthase promoter,the carboxylase small subunit promoter, the chlorophyll A/B bindingprotein promoter, and the promoters of the TR 1' and 2' agropine genes,J. Velten et al., EMBO J., 3, 2723-30 (1984).

Regeneration of plants transformed with Agrobacterium may be bytechniques of regeneration from plant protoplast, plant callus or planttissue known in the literature. See, e.g., D. L. Bidney et al.,Protoplasma, 117, 89-92 (1983) (cabbage); K. Glimelius et al.,Protoplasts (I. Potrykus ed., Birkhauser Verlag Basel), 64-65 (1983)(rapeseed); R. P. Hangarter et al., Plant Physiol., 65 761-67 (1980)(tomato) and R. Horsch et al., Science, 277, 1229-31 (1958) (tomato leafdisc method). Regenerated plants of normal morphology should beselected. Also, the ploidy of the regenerants is preferably the same asthat of the starting material; this can be verified by microscopy.

Inducible promoters can also be employed with the invention, inparticular in conjunction with the introduction of a dominant lethalmarker, which combination in effect becomes a dominant conditionallethal and which combination is to be considered a dominant conditionallethal marker for present purposes. For instance, a metallothioneinpromoter (of yeast or plant origin) is inducible with copper; the E.coli lac operon promoter is inducible with lactose or isopropylthio-beta-D-galactoside (IPTG); and the Tn10 tetracycline resistancepromoter is inducible with tetracycline. If a lethal marker is under thecontrol of an inducible promoter, it is conditionally lethal such thatapplication of the inducing agent will cause the marker to function as alethal agent.

Transformation of a plant in accordance with the method of the inventionpreferably results in the introduction of the foreign gene (the marker)as a single locus insertion in a fraction of the plants and morepreferably in at least a substantial fraction of the plants. Singlelocus insertion can be shown by screening of progeny in crossingexperiments, and single locus transformants can be selected. If thetransformation involves multiple (i.e., more than single) locusintroduction, single locus transformants can be obtained throughconventional breeding to segregate single locus progeny. That is, thetransformants can be outcrossed until monohybrid segregation ratios areobtained. In general, the Agrobacterium system transforms plants bysubstantially single locus insertion. See R. B. Horsch et al., Science,223, 496-98 (1984), and M. DeBlock et al., EMBO J., 3, 1681-89 (1984).Single locus transformants are heterozygous for the marker. The singleor multiple nature of the insertion can also be determined usingmolecular techniques such as Southern blotting.

Transformation of the plant also preferably results in the introductionof the foreign gene (the marker) into the host plant genome in asubstantially random fashion with respect to genetic location, therebyexpediting the task of selecting a plant with close linkage. That is,the probability of a given group of transformed plant cells having theintroduced gene closely linked to a particular locus, e.g., a malesterile locus, correlates with the extent of randomness of theintroduction. However, absolute randomness is not necessary for themethod of the invention. Rather, there need only be a sufficient numberof sites to which foreign DNA can be introduced to permit the productionof a plant with the introduced gene closely linked to a target locus(e.g., a male sterile locus). The extent of randomness in transformantscan be shown by mapping or by DNA analysis (e.g., Southern blot). In thecase of a male sterile locus, as stated, for many plants there will be anumber of such loci in a given plant species. The Agrobacterium systemis a preferred system for introducing DNA into a host plant in asubstantially random manner. See M. DeBlock et al., EMBO J., 3, 1881-89(1984).

In accordance with the invention a group of plant transformants isobtained containing the introduced marker. Depending on the size of thegroup, some of the plants may have the marker linked to a male sterilegene. Of these, some may have the marker gene closely linked (coupled)to a male sterile gene. Using the method of the invention linkage asclose as about 0.5 or even about 0.1 recombination unit is possible.Selection of a transformant with the desired linkage can be accomplishedby making a series of crossings of the transformants, followed byprogeny scoring (meaning testing or analyzing the progeny to determinesegregation patterns) in known ways in order to determine the relativeabundance of each phenotypic class and in order to select for a plantwith the marker gene linked to the male sterile locus. Assuming thatintroduction of loci occurs randomly on the genetic map, the likelihoodof obtaining a plant of a given closeness of linkage can be calculatedin view of the number of male sterile genes involved, the size of thegenome, and the number of transformants tested. The probabilitiesincrease with the number of transformants and the number of ms loci, anddecrease with departures from absolute randomness.

Linkage can also be determined using molecular techniques, e.g.,allozyme markers or restriction fragment length polymorphism markers, toidentify close-linkage transformants. See S. D. Tanksley, Plant Molec.Bio. Rptr., 1, 3-8 (1983) and references cited therein. Mapping atransformant with respect to an introduced marker and a known allozymemarker will reveal any linkage between the two. If there is linkage toan allozyme marker, and if the allozyme marker also has a known naturallinkage to a male sterile locus, the linkage (if any) between theintroduced locus and the male sterile locus can be determined. Forinstance, in tomato it is known that the allozyme locus Prx-2 is within1 unit of ms10 and the allozyme locus Skdh-1 is within 1 unit of ms32;C. M. Rick et al., Isozymes: Current Topics in Biological and MedicalResearch, Vol. II, 269-84 (1983). Thus, if the distance between anintroduced marker and the allozyme locus Skdh-1 is determined for atransformant, that distance, plus or minus 1 unit, is the distancebetween the introduced marker and ms32.

Selection of close linkage involves selection for linkage of markergenes and male sterile genes in coupling phase. That is, the dominantalleles of the male sterile locus and the marker locus are on the samechromosome, resulting in cosegregation upon crossing. If close linkageis found which is not in coupling, i.e., linkage in repulsion, linkagein coupling can be obtained by conducting crossings to select arecombinant, in ways known to those skilled in plant breeding.

Another type of linkage embraced by this invention is the use offlanking markers, that is, the use of two different markers on eitherside of the nuclear male sterile gene. This approach results incloseness of linkage which is superior (closer) than either of theindividual markers alone would provide for a given linkage distance.Thus, linkage of 10 units with each of two different flanking markers isapproximately equivalent to linkage of less than one unit with a singlemarker. This approach is particularly useful if tight linkage is notreadily obtained (e.g., due to departures from randomness) usingnon-flanking markers.

A transformed plant with introduced marker gene closely linked to atarget locus, once selected, can be used in breeding programs any timethere is a need or desire to follow the presence or absence of thetarget locus in a plant from generation to generation. In the case ofmale steriles, this method permits the identification of male sterileplants directly, without need for crossings to determine whether theplant is fertile or sterile. The method can also be useful in geneticstudies and breeding programs for marking or tagging other genes,particularly those difficult to score (e.g., disease resistance).

In the case of a male sterile locus as target locus, the method of theinvention embraces use of a plant as starting material which already hasa male sterile gene present either in homozygous or heterozygous form,e.g., a commercially available male sterile mutant line. The method alsoembraces the introduction of a male sterile gene into a male fertileplant (either homozygous or heterozygous for male fertility) prior to orsubsequent to transformation with a marker. Where the male sterile geneis introduced, the introduction may be accomplished using standardcrossing techniques known to plant breeders. For instance, crossing aplant (transformant or otherwise) which is homozygous for male fertility(Ms/Ms) with a plant which is homozygous for male sterility (m/ms)yields heterozygous plants (Ms/ms). A subsequent backcrossing of theheterozygous plants with the plant homozygous for male sterility (ms/ms)yields a 50:50 mixture of heterozygous (Ms/ms) and homozygous (ms/ms)plants. Crossing a plant (transformant or otherwise) which isheterozygous for male fertility (Ms/ms) with a plant which is homozygousfor male sterility yields a 50:50 mix of heterozygous (Ms/ms) andhomozygous (m-/m) plants.

Selection of a close-linkage transformant is made by appropriatecrossings and progeny scoring. A general procedure for this is describedbelow, with male sterile locus as target locus, with the understandingthat, as understood by those skilled in plant breeding, variations inthe steps or in the sequence of the steps can lead to comparableresults. The general procedure calls for transformation of homozygousmale fertile plants (Ms/Ms) as a first step. It should be understoodthat analogous procedures may be followed where the first step istransformation of a plant heterozygous for male fertility (Ms/ms) orhomozygous for male sterility (ms/ms). The procedure described belowuses as a marker the (dominant) conditional expressive antibioticresistance marker kanamycin resistance (KanR), but the procedure wouldbe analogous for other markers.

A plant homozygous for male fertility (Ms/Ms) is transformed to yield agroup of transformants which, assuming single locus transformation, areheterozygous for the introduced marker. If the marker is KanR, the plantis heterozygous for kanamycin resistance (KanR/--), or more strictly,hemizygous for KanR (when KanR is introduced by transformation into agiven chromosome, the homologous chromosome has only a deficiency, i.e.,the (--) allele has no physical existence and is defined operationally).Subsequently each transformant is tested to determine if the introducedgene (KanR) is present as a single locus. Each transformant is eitherself-crossed or crossed with a kanamycin sensitive (--/--) plant to makethis determination. Segregation of 3:1 (that is, 3 kanamycin resistantto 1 kanamycin sensitive) in self progeny or 1:1 in cross progeny isconsistent with single locus segregation. For instance, as shown in FIG.1 ("I. Dingle locus determination"), single locus introduction of themarker into the transformant (KanR/--) is shown if, in a cross of thetransformant with a line which is homozygous kanamycin sensitive plant(--/--), the progeny segregate 1:1 (resistant:sensitive). The larger thenumber of progeny scored, the greater is the meaningfulness of thesegregation data (the maximum being limited only by the number of seedsset and the number of crosses performed). At least forty progeny pertransformant are recommended. Transformants with multiple introducedloci are discarded, or backcrossed until single locus segregation isobtained.

Each transformant shown to be single locus is tested for close linkage.Each transformant (Ms/Ms) is backcrossed to a homozygous male sterile(ms/ms) line to yield first-generation progeny heterozygous for bothmarker and male fertility (this step is unnecessary if the transformantis already heterozygous for male fertility). The crossing step ispreferably repeated for each of a number of different male sterilelines, each line being homozygous for a different ms gene. The morelines that are used, the less important is any departure from randomnessof marker insertion with respect to the genetic map. Thefirst-generation progeny are backcrossed to the respective homozygousmale sterile lines to yield second-generation progeny which segregatefor both ms and marker loci, and which are scored for linkage. Linkagein the parent transformant is determined by the degree of associationbetween marker and ms loci in the second-generation progeny. Absolute(or near absolute) linkage in coupling is demonstrated for a giventransformant where the second-generation progeny are all fertileresistant or sterile sensitive (absolute linkage in repulsion is shownif the progeny are all fertile sensitive or sterile resistant). Forinstance, as shown in FIG. 1 ("II. Linkage determination" ), absolutelinkage is shown if, in a cross of a transformant heterozygous for malefertility and kanamycin resistance (Ms/ms;KanR/--) with a linehomozygous for male sterility and kanamycin sensitivity, progenysegregate only fertile resistant and sterile sensitive plants, with norecombinant plants (fertile sensitive or sterile resistant); non-linkageis shown by equal proportions of fertile resistant, fertile sensitive,sterile resistant, and sterile sensitive. The fewer fertile sensitiveand sterile resistant plants (i.e., recombinant plants) relative tofertile resistant and sterile sensitive plants, the closer is thelinkage. Again, the larger the number of progeny scored, the greater thereliability of the linkage data. Where sufficiently close linkage isshown, the heterozygous male parent which is the parent of theclose-linkage second-generation progeny is selected for use in hybridbreeding (or for crossing into other breeding lines or varieties of thespecies). If desired, verification of the close linkage may be made byadditional crossings.

If the above procedure is followed for a plant with a typical totalgenomic length of approximately 1000 recombination units (e.g., tomato,barley, corn) and for which, e.g., five independent male sterile lociare used (i.e., five different lines, each with a different male sterilelocus), it is necessary to screen approximately 299 transformantscarrying a single marker locus in order to attain an approximate 95%probability of finding a plant having an introduced marker locus withinone map unit of a male sterile locus, assuming random integration. Toachieve a 95% probability for other levels of closeness, it is necessaryto screen approximately 99 plants for a closeness of 3 units; 59 plantsfor a closeness of 5 units; 29 plants for a closeness of 10 units; 14plants for a closeness of 20 units; and 11 plants for a closeness of 25units. Each of the transformants (e.g., each of the 299) is subjected tobackcrossing and progeny scoring as described above in order to selectfor the desired closeness of linkage. Once the close-linkage plant isobtained, the male fertile phenotype can be predicted by the presence ofthe marker, with an accuracy proportional to the degree of linkage.

A general procedure is described below for use in hybrid breeding of aclose-linkage plant made in accordance with this invention, again withthe understanding that variations in the steps or in the sequence of thesteps, as understood by those skilled in plant breeding, can lead tocomparable results. The procedure is described using the (dominant)conditional expressive antibiotic resistance marker KanR, again with theunderstanding that the procedure is analogous for other markers.

A homozygous male sterile plant (ms/ms) is crossed with an inbred line(Ms/Ms) destined to be the female parent in hybrid production. The crossyields first-generation (F1) progeny heterozygous for male fertility(Ms/ms). The F1 progeny are backcrossed to the inbred and from thefirst-generation backcross progeny (BC1) heterozygous male fertile(Ms/ms) plants are selected by progeny testing. The selected BC1 progenyare backcrossed several times to the inbred line to maximize selectiveintroduction of the male sterile gene into the inbred's genome. Lastly,heterozygous (Ms/ms) progeny are self-crossed to produce a homozygous(ms/ms) inbred line.

In parallel, an analogous procedure is carried out for a close-linkageplant, homozygous for male fertility (Ms/Ms) and marker (KanR/KanR).(The close-linkage plant as originally selected can be converted to thishomozygous form by appropriate crossings and selections, as explainedpreviously.) The close-linkage plant is crossed with the same inbredline as above (ultimate female parent), the F1 progeny are backcrossedto the inbred, and plants heterozygous for marker (KanR/--) areselected. The selected BC1 progeny are backcrossed several times to theinbred line to maximize selective introduction of the closely linkedmarker gene into the inbred's genome. Heterozygous (KanR/--) progeny areselfed to produce a homozygous (KanR/KanR) inbred.

The two inbreds resulting from the above, one carrying the malesterility allele and the other carrying the marker closely linked to themale fertility allele, but otherwise nearly isogenic, are crossed togenerate progeny from which a maintainer line is selected. Theclose-linkage maintainer line is heterozygous for male fertility (Ms/ms)and for closely linked marker (KanR/--).

The maintainer line is backcrossed to a homozygous male sterile andkanamycin sensitive line (ms/ms;--/--), e.g., the female parent of themaintainer line, to yield a mixture of, first, plants homozygous forboth male sterility (ms/ms) and absence of marker (--/--) and, second,plants heterozygous for male fertility (Ms/ms) and closely linked marker(KanR/--). The mixture of plants is planted as the female parent forhybrid breeding, e.g., as the female row in a hybrid crossing row.Testing of all the plants in this row for presence of the marker, whichin effect is a test for male fertility, permits removal of male fertileplants at an early stage.

Identification and removal of the male fertiles from the aggregation ofmale fertiles and male steriles resulting from crossing of themaintainer line is accomplished in different ways depending on themarker which is closely linked. In each case, however, removal occurs ata pre-anthesis stage, preferably at the seed or seedling stage. If themarker is a visible expressive marker, the presence or absence of color(depending on whether the marker is dominant or recessive) is the basisfor identifying and removing male fertiles. If the marker is anon-visible but assayable expressive marker, an appropriate assay isconducted, e.g., on a piece of leaf. If the marker is a conditionalmarker, e.g., chromogenic, resistance, or dominant conditional lethal,the chemical agent in question may be applied, e.g., in a spray directedto either part of the plant or all of the plant, at a dosageindependently determined to be effective given the level of expressionof the marker in the close-linkage plant. In the case of a chromogenicmarker the presence or absence of developed color (depending on whetherthe marker is dominant or recessive) is the basis of identification. Inthe case of resistance markers, which are normally dominant, absence ofobservable damage to the plant tissue to which the chemical is appliedis the basis of identification of male fertiles. For KanR, the test maybe carried out by growth of an excised plant part, e.g., leaf or stem,in the presence of the antibiotic in appropriate dosage in sterileculture. Tissue from male fertiles will grow; tissue from male sterileswill not. Once identified as male fertiles based upon chromogenic orresistance markers, male fertiles are removed, e.g., by hand ormechanically in the field or, e.g., by optical scanner and sorter ifidentification is by color at the seed level. In the case of dominantconditional lethal markers, application of the chemical, which may bebest accomplished by spraying the entire plant or field, automaticallyand selectively weakens or destroys the male fertiles, preferablywithout need for physical removal.

Removal of male fertile plants results in an essentially pure set ofmale sterile plants. The essentially pure set of male sterile plants isused as the female parent stand in hybrid crossing. This stand isinterplanted with stands of the intended male parent and hybrid seedresults from pollination by wind or insects. Alternatively, pollinationcan be by hand. Selectively harvesting the female parent rows yieldsessentially pure hybrid seed. The maintainer line, which is heterozygousfor both male fertility and marker, may be maintained indefinitely byappropriate crossings and selections, as described above and asunderstood by those skilled in plant breeding.

EXAMPLES

In the following examples, reagent materials are commercially available,unless otherwise specified. Enzymes used in the cloning procedures areavailable from commercial sources. Restriction endonuclease reactionsare carried out according to manufacturer instructions. Unless otherwisespecified, the reaction conditions for other reactions are standardconditions used in the art, as described, for example, in T. Maniatis etal., Molecular Cloning, Cold Spring Harbor Laboratory, New York (1982)(referred to herein as "Maniatis"); R. W. Davis et al., AdvancedBacterial Genetics, Cold Spring Harbor Laboratory, New York (1980); D.A. Evans et al., Handbook of Plant Cell Culture, Vol. 1, ed., MacMillan,New York (1983); and J. H. Miller, Experiments in Molecular Genetics,Cold Springs Harbor Laboratory, New York (1972) (referred to herein as"Miller").

Plasmids pBR322 and pUC8 are multicopy plasmid vectors, availablecommercially, Bolivar et al., Gene, 2, 95-113 (1977); J. Veira et al.,Gene, 19, 259-268 (1982) (referred to herein as "Veira"). Plasmid DNAwas prepared by alkaline extraction according to H. C. Birnboim et al.,Nucleic Acids Res. 7, 1513-1523 (1979) (referred to herein as"Birnboim"), unless stated otherwise. All ligation reactions wereperformed in a final volume of 20 microliters (ul). at 15° C. for 16hours with 400 units T4 DNA ligase. Transformations of E. coli strainswere performed according to M. Dagert et al., Gene, 6, 23-28 (1979)(referred to herein as "Dagert").

M9, minA, and LB media are described by J. H. Miller, Experiments inMolecular Genetics, Cold Spring Harbor Laboratory, New York (1972). MSmedium is described by Murashige et al., Physiol. Plant, 15, 473-97(1962) (referred to herein as "Murashige"). Micrograms are referred toherein as ug; microliters are referred to herein as ul; and nanogramsare referred to as ng.

EXAMPLE 1 Introduction of Resistance Conditional Expressive Marker(KanR) into Tomato Hybrid A. Preparation of Agrobacterium tumefaciensC58Cl/pGV3850 kanR

Agrobacterium tumefaciens C58Cl/pGV3850 kanR contains acceptor Tiplasmid pGV3850 kanR which can (1) transfer T-DNA to plant cells; (2)"accept" intermediate vectors having homology to pBR322 sequencescarried on pGV3850 kanR; and, (3) direct nopaline synthesis and conferkanamycin resistance upon transfer of T-DNA to plant cells. See P.Zambryski et al., European Patent Application No. 116,718 (publishedAug. 29, 1984) for a general description of such plasmids and thisapproach.

Plasmid pGV3850kanR was constructed using pGV3850 (P. Zambryski et al.,EMBO J., 2, 2143-50 (1983)) and pLGV232neo (generally available).Plasmid pLGV232neo is a derivative of pLGV23neo (Herrera-Estrella etal., EMBO J., 2, 987-95 (1983)) constructed by introduction of the ocs(octopine synthase) 3' tail fragment to the Tn5 SmaI site in pLGV23neo.Plasmid pLGV232neo can be reconstructed from (1) plasmid pLGV23neo and(2) plasmid pSS155 (S. Schweitzer et al., Plasmid, 4, 196-204 (1980)),containing a PvuII fragment with an ocs polyA signal, by ligating thefragment to the SmaI site of pLGV23neo.

The preparation of pGV3850kanR was carried out as follows. A BglIIlinker was introduced into the PstI site of pBR322 to yield a pBR322derivative by digestion of pBR322 with PstI, treatment with T4 DNApolymerase (Maniatis, supra), and ligation to BglII linker (commerciallyavailable). A BglII digest of the pBR322 derivative was prepared.pGV232neo was partially digested with BclI and then digested with BamHIto yield a pGV232neo digest. The digest of the pBR322 derivative wasligated to the digest of pGV232neo. Transformation of E. coli strainJM83 (Veira, supra) was carried out using standard procedures (Maniatis,supra; Dagert, supra). Transformants were selected on plates containingLB medium supplemented with tetracycline (15 ug/ml) and kanamycin (50ug/ml). A plasmid clone was obtained, with restriction sites in thesequence BglII/Bam--BclI BclI/BglII--HindIII--Bam, by screeningtransformants based on restriction patterns (Birnboim, supra). Theplasmid clone was digested with HindIII and Bam. The plasmid clonedigest was ligated into pRK404 digested with HindIII and Bam. PlasmidpRK404, a derivative of pRK290 (G. Ditta et al., PNAS, 77, 7347-51(1980)), is generally available. The resulting plasmid, pJJ108, wasmobilized into strain C58Cl/GV3850 using pRK2013 (G. Ditta et al., PNAS,77 7347-51 (1980)) as a helper. Exconjugants resulted from either singleor double crossover events, based on pBR322 sequences present in pGV3850and in the plasmid mobilized into GV3850. Double crossover events wereselected by mating conjugants with BH101/pPHlJI (P. R. Hirsch et al.,Plasmid, 12, 139-41 (1984)) and selecting gentamycin resistance andkanamycin resistance (pPHlJI is incompatible with pRK404 and, byselecting for pPHlJI with gentamycin, pRK404 single recombinants aredriven out and only double recombinants survive). The resultant strain,Agrobacterium tumefaciens C58Cl/pGV3850kanR was used to introduce theKanR marker into plants.

B. Preparation of Plants

L. esculentum×L. pennellii seeds (generally available) weresurface-sterilized by treatment with 70% ethanol/water for 1-2 minutes,followed by 10% commercial bleach for 30 minutes and rinsed with sterilewater three times for ten minutes. These were germinated in sterileplastic containers on hormone-free MS medium under sterile conditions ina culture room or incubator (16 hour day, 5000 lux white fluorescentlight, 27° C.). Seedlings were decapitated by transversely cutting theepicotyl, and tops rooted in fresh medium (plants can be maintained inculture for long periods of time by periodically passaging the tops inthis manner). Standard aseptic techniques were used for all steps inshoot and tissue culture.

C. Transformation by Stem Inoculation

The following procedure was carried out generally in accordance with P.Zambryski et al., European Patent Application No. 116,718 (publishedAug. 29, 1984).

Epicotyls from seedlings or rooted shoots were cut transversely intosegments about 1 cm long, which were held upright in normal or invertedorientation by inserting into hormone-free MS solid medium. The uppercut surface was inoculated with a fresh Agrobacterium C58Cl/pGV3850kanRculture which had been prepared as described above and grown on M9+0.4%sucrose plates (Miller, supra), supplemented with kanamycin sulfate (100mg/l). Inoculation was done with a sterile platinum loop or toothpickwith an amount of cells sufficient to cover the cut surface. These wereincubated three days in an incubator (same as above except 500 lux),then the inoculated surface was excised with a scalpel as a 1-3 mm thickslice. This slice was placed upright on selective callus medium (MSmedium supplemented with 2% sucrose, B5 vitamins [nicotinic acid (100mg/l), thiamine (1 g/l), pyridoxine (100 mg/l), and myo-inositol (10g/l); Gamborg et al., Exp. Cell Res., 50, 151-58 (1968)], zeatin (0.5mg/l), 1-naphthaleneacetic acid (1 mg/l), carbenicillin 250 mg/l, andkanamycin sulfate (50 mg/l). Four to six weeks after inoculation,kanamycin resistant calli appeared on the inoculated surfaces of slices.

D. Regeneration

Resistant calli were removed with a scalpel and transferred to freshmedium for several weeks until large enough (4-10 mm diameter) fortransfer to shooting (regeneration) medium (MS supplemented with 2%sucrose or glucose, B5 vitamins (as above), and zeatin (2 mg/l)) under5000 lux. Callus was transferred to fresh shooting medium at three weekintervals until shoots formed (0-3 transfers). Shoots were rooted under5000 lux in the following rooting medium: NH₄ NO₃ (400 mg/l), KNO₃ (1010mg/l), MgSO₄.7H₂ O (370 mg/l), and CaCl₂.H₂ O (440 mg/l) in 0.8% agarsupplemented with B5 vitamins (as above), 3% sucrose, andindole-3-butyric acid (0.15 mg/l). After rooting (2-8 weeks) plants wereremoved from agar, planted in soil, and placed under mist in agreenhouse. 33 regenerated plants (transformants) were obtained.

E. Characterization of Transformants 1) Ploidy

Basal shoots were excised from regenerated plants and rooted withRootone in soil. To determine ploidy, each of the regenerants wascrossed to diploid L. esculentum (crosses between polyploid tomatoes anddiploid tomatoes do not produce viable seed, i.e., only diploidtransformants produce seeds when crossed to a diploid). Of 33transformants, 12 were diploid and 21 were polyploid.

2) Kanamycin Resistance

Eleven of the twelve diploids were kanamycin resistant as judged by theability of their shoots to root in the presence of kanamycin sulfate (10mg/l) on MS agar and by the ability of leaf segments to form callus oncallus medium supplemented with kanamycin sulfate (100 mg/l).

3) Nopaline Synthesis

Eight of the KanR plants produced nopaline as assayed by paperelectrophoresis of extracts (L. A. Otten et al., Biochim. Biophys. Acta,527, 497-500 (1978)).

4) Southern Blot Analysis

Southern blot analysis (Maniatis, supra) of the eleven diploid KanRtransformants was used to determine the number of copies of the rightborder (RB) region of the T-DNA inserted into the genome of eachtransformant. Right border fragments were detected in the Southern blotby hybridization to homologous P32 labelled T-DNA fragments (ClaI+PvuIIfragment isolated from pT37H23 (A. Depicker et al., J. Molec. andApplied Genet., 1, 561-73 (1982)). Six of the eleven transformantspossessed a single right border fragment; five possessed two or moreright border fragments. The number of right border fragments isindicative of the maximum number of introduced genetic loci determiningkanamycin resistance in the transformant. Thus, at least six of theeleven transformants tested were single locus KanR transformants.

Each of the eleven transformants was also analyzed by Southern blot inorder to compare the sizes of different right border fragments. It wasfound that each right border fragment was distinct in size, leading tothe conclusion that each was the result of a distinct insertion event.This is evidence that each introduced KanR gene was present at adifferent site in the plant genome, a finding consistent with theexpectation that the T-DNA integrated substantially randomly in thegenome.

EXAMPLE 2 Selection of Close-Linkage Plants

Seven diploid KanR transformants from Example 1 were selected forlinkage analysis using allozyme markers of known chromosomal position inthe tomato genome. The group of seven transformants included the sixtransformants determined by Southern analysis to be single copytransformants. The seven transformants were subjected to outcrossingswith a commercial line of L. esculentum and progeny scoring to determinepossible linkage relationships between the introduced KanR markers and anumber of known allozyme markers.

Each transformant was found to show linkage between introduced KanR andan allozyme locus known to be positioned on a particular chromosome.Introduced loci showed genetic linkage to allozyme markers onchromosomes 1, 2, 8, 10 and 12. Male sterile loci are known to exist onfour of these five chromosomes: chromosome 1 (ms6, ms32); chromosome 2(ms2, ms5, ms10, ms15); chromosome 8 (ms8, ms17); and chromosome 10(ms31). See C. M. Rick, Report of the Tomato Genetics Cooperative, 20,2-17 (1980) with respect to ms loci in tomato. A transformant whichshowed genetic linkage between KanR and allozyme markers on chromosome 2(transformant A181, one of the single locus transformants) evidencedlinkage between KanR and ms loci on chromosome 2, as explained below.

The allozyme loci Est-7 (esterase-7) and Prx-2 (peroxidase-2) are knownto be positioned on chromosome 2. The linkage relationships betweenthese allozyme loci and the ms loci on chromosome 2 are known to be asfollows: ms10 is 1 map unit (centimorgan) from Prx-2, with the direction(i.e., which "side" of Prx-2) being unknown; m15 is 10-12 map units fromPrx-2; ms2 is 17-19 map units from Prx-2 and on the same "side" of Prx-2compared to ms15; and ms5 is 13 units from ms2, direction unknown. Thelocus Est-7 is 12 units from Prx-2, on the opposite "side" of Prx-2compared to ms15, ms2 and ms5. See C. M. Rick et al., Isozymes: CurrentTopics in Biological and Medical Research, 11, 264-84 (1983); and C. M.Rick, Report of the Tomato Genetics Cooperative, 20, 2-17 (1980).

Allozyme mapping of 71 progeny of A181 showed the KanR introduction tobe positioned 25 map units from Prx-2 and 32 map units from Est-7. Thatis, the introduced KanR locus was on the same "side" of Prx-2 as ms15,ms2 and ms5. Based on the known linkage distances between Prx-2 and msloci set forth above, the linkage measurements for the introduced KanRlocus evidenced linkage of KanR to m10 (linkage distance 24-26 units),ms15 (linkage distance 13-15 units) and ms2 (linkage distance 6-8units). (Linkage of KanR to ms5 was either 5-7 units or 19-21 units,depending on which "side" of ms2 the ms5 locus maps.)

This transformant, with KanR linked to a male sterile locus, can besubjected to appropriate crossings with a line homozygous for thedesired closely linked male sterile locus, as previously described, foruse in hybrid breeding.

EXAMPLE 3 Introduction of Dominant Conditional Lethal Marker (iamH) intoTobacco A. Preparation of Agrobacterium

The gene (iamH) for the enzyme indole acetamide hydrolase was obtainedfrom pGV0153 (De Vos et al., Plasmid, 6, 249-53 (1981)) as follows (seeFIGS. 2-4). Two ug of pGV0153 DNA were digested with 2 units BamHI andelectrophoresed in 1% agarose. Agarose containing the Bam 8 fragment wascut out of the gel and the DNA recovered by electroelution (Maniatis,supra). The Bam 8 fragment (corresponding to the left part of TL-DNA ofthe octopine plasmid pTiB6S3) contained the iamH gene.

Five ug pUC8 DNA was totally digested with 5 units BamHI. 0.2 ugBamHI-cleaved pUC8 DNA was ligated to the recovered Bam 8 fragment. E.coli JM83 (Veira, supra) was transformed with this mixture and cellswere plated on LB medium supplemented with ampicillin (100 m /l) andbromo-chloro-indolyl-B-D-galactoside (40 mg/l). Two white colonies werescreened by alkaline plasmid extraction and BamHI digestion of theisolated DNA. One clone contained a plasmid with Bam 8 inserted at theBamHI site of pUC8 and was designated pAGS751. Orientation of the Bam 8fragment in pAGS751 relative to pUC8 was determined by SalI and SmaIdigestions. Plasmid pAGS751 is diagrammed in FIGS. 2 and 3. In FIG. 2,"H" refers to HindIII; "Bam" refers to BamHI; "Sma" refers to SmaI; and"Sal" refers to SalI sites. The numbers 1, 2, 5 and 7 refer to TL genes,with "2" being the iamH gene. The symbols in FIG. 3 are the same as inFIG. 2, except "Hin" refers to HindIII.

2 ul pAGS751 DNA (from alkaline extraction) was digested with BamHI andHindIII. 2 ug pBR322 DNA was totally digested with HindIII and treatedwith 25 units calf alkaline phosphatase. 0.2 ug CAP-treated pBR322 DNAwas phenol extracted and ligated to pAGS751 BamHI and HindIII cleavedDNA in 20 ul. E. coli MM294 (M. Meselson et al., Nature, 217, 1110(1968)) was transformed with this mixture. Cells were plated on LBmedium supplemented with ampicillin (100 mg/l). Plasmid DNA extractionsof twenty-four colonies were digested with HindIII and electrophoresedto identify those in which the 2.3 kb HindIII fragment was present. Twocolonies (designated pAGS752 and pAGS753) carried this fragment and thevector pBR322, and were cleaved with EcoRI and SmaI to determine theorientation of HindIII 2.3 relative to the vector. pAGS753 carriedfragments of 5.1 kb, 1.1 kb, and 0.5 kb, indicating the orientationshown in FIG. 3.

2 ul pAGS751 DNA (from alkaline extraction) was totally digested withSmaI. The resulting digestion was ligated and used to transform MM294.Cells were plated on LB medium supplemented with ampicillin (100 mg/l).Alkaline extraction was used to isolate DNA from 36 colonies and one(designated pAGS754) was identified by digestion with SmaI and BamHI asa deletion removing SmaI fragments 2.6 kb and 1.8 kb in size. PlasmidpAGS754 is diagrammed in FIG. 2 and 3.

pAGS753 carried the complete coding sequences of iamH and 3' sequenceswhich carry the presumed polyadenylation sequences, but not sufficientsequence 5' to the coding sequence to provide a promoter for itstranscription. pAGS754 carried complete 5' sequences but not the 3' halfof the coding sequences. In order to reconstruct a functional iamH genefrom these plasmids, 8 ul DNA (alkaline extraction) of pAGS753 and 2 ulDNA of pAGS754 were digested with SmaI and BamHI, and these were ligatedand used to transform MM294. Cells were plated on LB medium supplementedwith ampicillin (100 mg/l). Plasmid DNA was isolated from forty-eightcolonies by alkaline extraction and intact plasmids were screened byelectrophoresis for plasmids larger than pAGS753. Six were found andthese were screened by electrophoresis of EcoRI digestions. Two werefound which carried the 1.1 kb EcoRI fragment of the iamH gene. Thesewere analyzed further by electrophoresis of digestions with BglII andHindIII. Comparison with similar digests of pAGS751 was used todemonstrate that these two plasmids (designated pAGS755 and pAGS756)contained fragments of an intact iamH gene (diagrammed in FIGS. 2 and3).

pAGS755 and pAGS756 were each separately ligated to a binary vectordesignated pAGS113. See Hoekema, et al., Nature, 303, 179-80 (1983)regarding binary vector systems and plasmids of this type. pAGS113 is aderivative of pRK404 that carries the right and left borders of TL andthe nos-nptII-ocs chimeric gene of pLGVneo2103 (generally available).Two unique restriction sites, BamHI and ClaI, lie between the TL bordersof pAGS113. Plasmid pAGS113 is shown as a segment of pAGS757, pAGS758and pAGS759 in FIG. 4. The plasmid is derivable from pAGS757 (on depositwith the ATCC as ATCC No. 53066, see below) by cutting with ClaI andligating to circularize, as will be understood by those skilled in thisarea. In FIG. 4, "C" refers to ClaI; "H" refers to HindIII; "LB" and"RB" refer to left and right borders, respectively; and "2" refers tothe iamH gene.

The transfer of pAGS755 to pAGS113 was accomplished as follows. 5 ng ofpAGS755 and 250 ng of pAGS113 were digested with ClaI and ligated. Theligation was used to transform MM294 and cells were plated on LBsupplemented with tetracycline (20 mg/l). The transfer of pAGS756 topAGS113 and subsequent transformation and plating were carried out inthe same way. Six colonies from the pAGS755 ligation and three from thepAGS756 ligation were screened by HindIII digestion of alkalineextraction plasmid DNA and compared to pAGS755 and pAGS756. Onederivative of pAGS756 (designated pAGS759) was a fusion of pAGS113 andpAGS756, and two derivatives of pAGS755 (designated pAGS757 and pAGS758)were fusions of pAGS113 and pAGS755. The orientations of pAGS757 andpAGS759 were the same; that of pAGS758 was the opposite.

Transfer to Agrobacterium was accomplished as follows. RecipientAgrobacterium tumefaciens strain LBA4404 (Hoekem et al., Nature, 303,179-80 (1983)) was grown 40 hours in min A medium at 28° C. An E. colistrain carrying helper plasmid pRK2013 and E. coli donor strainsMM294/pAGS757, MM294/pAGS758 and MM294/pAGS759 were grown overnight inLB at 37° C. In three separate mixings, one ml of recipient and one mlof helper culture were mixed with one ml of one of the three donors.Three mixtures, one for each donor, were centrifuged. Pellets were takenup in 100 ul 10 mM MgSO₄, and the resulting suspensions spotted on LBplates which were then incubated at 28° C. overnight to allow mating tooccur. Cells were washed off each plate into 2 ml MgSO₄. 0.1 ml of thesesuspensions were plated on LB plates supplemented with rifampicin (100mg/l) and tetracycline (2.5 mg/l) and incubated at 28° C. for fortyhours. Colonies were streaked out on M9 plates supplemented with sucrose(0.4%) and tetracycline (2.5 mg/l). The resultant A. tumefaciens cells,one isolate from each of the matings, were designated A. tumefaciensLBA4404/pAGS757, LBA4404/pAGS758 and LBA4404/pAGS759, respectively.(LBA4404/pAGS757 has been deposited as ATCC No. 53066). A. tumefaciensLBA4404/pAGS113 was prepared in the same fashion as above for use as acontrol.

B. Preparation of Plant Material

N. tabacum cv. Wisconsin 38 was maintained as an axenic culture on theagar solidified medium MS of Murashige (supra). Apical meristems (1 cm)were excised at 5-7 week intervals, concurrent with the use of the leafmaterial in protoplast isolation, and placed individually in 3×4 inchMagenta cups containing 50 ml media. The cultures were maintained at 28°C. under "cool white" fluorescent light at 6000 lux, 16 hr/day.

Protoplasts were isolated from expanded leaves. Between 0.5 and 1.0 gmof tissue (fresh weight) was finely divided with a surgical blade in 20ml of K3 medium (J. I. Nagy et al., Pflazerphysiol., 78, 453-55 (1976))which contained Cellulase (Cellulysin or Onozuka), 1%; Macerase(Onozuka), 0.1%; Mes buffer (Sigma), 0.1%; and, the elevatedphytohormone regime of Caboche (M. Caboche, Planta, 149, 7-18 (1980)) atpH 5.6.

Cell wall digestion proceeded overnight (16-18 hours) at 29° C. indarkness with gentle gyrotory shaking (approx. 20 rpm). The digest waspoured through a funnel containing four layers of cheesecloth and thecheesecloth was rinsed with K3 media and expressed into Babcock bottles.The bottles were centrifuged 10 minutes at 100 g's. The floatingprotoplasts were collected with a 1 ml serological pipette, resuspendedin a fresh bottle of K3 media and centrifuged again. The protoplastswere collected as before and placed in a known volume of K3 media withglucose (0.4M) replacing sucrose and again the elevated phytohormoneregime. The protoplasts were then counted in a hemocytometer and thevolume adjusted to provide a final density of 10⁵ protoplast/ml. Theprotoplasts were cultured in 5 ml aliquots in 100 mm×20 mm "tissueculture treated" petri dishes in darkness at 27° C. for 30 hours priorto cocultivation.

C. Cocultivation of A. tumefaciens With Plant Cells

A single bacterial colony of A. tumefaciens LBA4404/pAGS757 prepared asabove was suspended in 5 ml minA medium and grown overnight at 27° C. ona roller drum at 40 rpm. An additional 9 hours growth period, afterdilution, produced approximately 3 doublings and an appropriatebacterial growth phase/culture density. Microliter amounts of thesuspension were then mixed with plant cells prepared as above to yield amultiplicity of 50 bacteria/protoplast (0.D. 550 nm 0.8, correspondingto 10⁹ bacteria/ml). Cocultivation proceeded for 66 hours at 24° C. and500 lux. Four days after protoplast isolation the cocultivation wasterminated by dilution to 2×10⁴ protoplast equivalents/ml with K3 mediumcontaining cefotaxime (500 ug/ml) and the decreased phytohormone regimeof Caboche (M. Caboche, Planta, 149, 7-18 (1980)). Thereafter the mediawas replaced at 3 and 4 day intervals (twice a week) by pelleting thecells by centrifugation at 50-70 g's for 5 minutes. The osmoticum wasreduced by 0.05M at each interval by mixing, first, K3 media with anappropriate amount of Media C (J. R. Muller et al., Physiol. Plant, 57,37-41 (1983)) containing sucrose 0.1M, mannitol 0.2M, and later Media Cwith MS. After three such replacements, cefotaxime could be eliminatedfrom the media without danger of continued bacterial proliferation. Thisentire procedure was also carried in parallel with A. tumefaciensLBA4404/pAGS758 and LBA4404/pAGS759, and with the control A. tumefaciensLBA4404/pAGS113.

Transformed plant cells were selected in each of the parallelexperiments as follows. Antibiotic resistance in the plant cells wasselected by replacing with medium containing kanamycin (50 ug/ml)starting 8 days after protoplast isolation. Transformed calli becamevisible approximately 21 days after protoplast isolation and the culturedensity was reduced (4×) to 5×10³ starting protoplast equivalents perml. At day 25, a further dilution was required to facilitate counting ofthe transformed calli. At this time the density was reduced (5×) to 10³protoplast equivalent per ml and the medium, MS, was solidified usingagarose (Sigma type VII) at a final concentration of 0.25%. Antibioticresistant calli were counted at day 30.

D. Regeneration of Plants

Regeneration of whole plants was carried out substantially in accordancewith the procedures described in P. Zambryski et al., EMBO J., 2,2143-50 (1983), and in P. Zambryski et al., European Patent ApplicationNo. 116,718 (published Aug. 29, 1984). Transformants were potted insoil.

E. Characterization of Transformants 1) Conditional Lethal Phenotype

After potting in soil, the transformants were acclimated to thegreenhouse for one week at which time all appeared to be healthy andactively growing. In the first experiment, four plants each fromtransformations with A. tumefaciens LBA4404/pAGS757, LBA4404/pAGS758,LBA4404/pAGS759 and LBA4404/pAGS113 were sprayed with a 1 mg/ml solutionof naphthalene acetamide (NAM), two plants each, or naphthalene aceticacid (NAA), two plants each, in 20% ethanol, 0.8% Tween 80. Each plantwas sprayed to wet completely all upper leaf surfaces (3 to 5 ml perplant). Plants were sprayed within one hour of dusk to minimizeevapotranspiration. Four days after spraying, all plants sprayed withNAA showed severe epinasty and had stopped growth; all these plantseventually died. Plants transformed with pAGS113 (control) and sprayedwith NAM showed epinasty at the outset, but four days after sprayingexhibited reversal of epinasty and resumed growth. The NAM controlplants recovered completely. NAM-sprayed plants transformed withpAGS757, pAGS758, or pAGS759 exhibited a range of phenotypes betweenthese two extremes. One of the pAGS757 plants was indistinguishable fromthe pAGS113 plants, while the other exhibited intermediate sensitivity,recovering five weeks after spraying. Of the two pAGS758 plants, oneresponded the same as pAGS113 plants, while the other responded the sameas an NAA sprayed pAGS113 plant, i.e., exhibited conditional lethality.One pAGS759 plant in this experiment responded exactly as pAGS113plants; the other displayed weak sensitivity to NAM for nine days, butat nineteen days was indistinguishable from pAGS113 plants.

In a second experiment, two pAGS758 plants were sprayed with NAA; andfour pAGS113 plants, two pAGS757 plants, three pAGS758 plants, and onepAGS759 plant were sprayed with NAM. In this experiment, plants hadeleven days to acclimate and were larger than in the previousexperiment. Consequently, the two NAA sprayed plants showed slightlyless sensitivity than in the previous experiment and were still alive,though very weak, at 31 days. The four pAGS113 plants respondedsimilarly to the previous experiment. At 31 days, one pAGS757 and onepAGS758 plant were dead, i.e., responded even more severely than did NAAsprayed plants, one pAGS757 plant was very weak and though not dead wasweaker than NAA plants, one pAGS758 plant was slightly sensitive(intermediate between NAA and NAM control plants), and the last pAGS758and the only pAGS759 plant were equivalent to the pAGS113 plants. Thusit was possible to identify plants, among transformants carrying theiamH gene, that were at least as sensitive to NAM as to NAA. The resultsof the two experiments are shown in Table I. The numbers in the body ofthe Table indicate the number of plants having a given response rating(a dash means zero).

                  TABLE I    ______________________________________              Response Rating:              1      2     3         4   5    ______________________________________    Experiment I    A. NAA spray    pAGS    113     --       --  --      --  2            757     --       --  --      --  2            758     --       --  --      --  2            759     --       --  --      --  2    B. NAM spray    pAGS    113     2        --  --      --  --            757     1        --  1       --  --            758     1        --  --      --  1            759     1        1   --      --  --    Experiment II    A. NAA spray    pAGS    758     --       --  --      2   --    B. NAM spray    pAGS    113     4        --  --      --  --            757     --       --  --      1   1            758     1        --  1       --  1            759     1        --  --      --  --    ______________________________________     Response Ratings:     1  Minimum effect; full recovery.     2  Very slight sensitivity; full recovery in two weeks.     3  Intermediate sensitivity; little or no recovery evident until 4 weeks.     4  Very sensitive; very weak but alive at 4 weeks.     5  Maximum sensitivity; dead at 4 weeks.

To recover plants that were maximally sensitive to NAM, plants weredecapitated after potting to soil, and after side shoots appeared theywere excised and rooted using rooting powder (Rootone). After rootingthey were transferred to soil and, after acclimation, sprayed with NAA.The parent plant or one of the cuttings was maintained in order torecover self-cross and backcross seed. For testing response to NAM,rooted cuttings were kept in a growth chamber with high illumination 14hour days 30° C. C/10 hour nights 20° C. The effect of NAM was lesssevere in this experiment than in the previous two experiments.Duplicate cuttings were sprayed to control for effects of differentialdegrees of rooting. Two pAGS757 transformants, four pAGS758transformants, and two pAGS759 transformants were sprayed in duplicatealong with three pAGS113 plants. Two pAGS758 transformants were severelyinhibited and not growing at 20 days. Other plants displayed either thesame effects as pAGS113 controls (which responded as in previousexperiments, only more mildly) or an intermediate effect. Variability inNAM phenotype among individual transformants may be related to variationin the level of expression of the introduced genes in the transformants(e.g., R. Horsch et al., Science, 227, 1229- 31 (1985)). Clones of eachtype were grown to flowering and selfed and backcrossed to obtainprogeny. Progeny can be analyzed for heritability of the NAM phenotypeand segregation of T-DNA.

2) Segregation Data

Segregation analysis to determine extent to which introduction of thedominant conditional lethal marker in a given transformant is singlelocus, random and stable can be conducted with plants reproduced (e.g.,vegetatively prior to lethality test) from transformants displaying theconditional lethal phenotype. Single locus plants can be used forselection of close-linkage plants, e.g., in accordance with Example 2.

EXAMPLE 4 Introduction of Resistance Conditional Expressive Marker(kanR) into Tobacco A. Transformation of Plants

Wisconsin 38 tobacco (Nicotiana tabacum) protoplasts, prepared as inExample 3, were transformed by cocultivation as in Example 3, but usingA. tumefaciens strain C58Cl/GV3850kanR of Example 1, to yieldtransformants which were regenerated as in Example 3.

B. Progeny Testing

The plants so obtained were self-pollinated and backcrossed to Wisconsin38. Progeny were scored for kanamycin resistance by surface sterilizing,germinating on filter paper in

the dark for three days, growing under 500 lux for 1-2 weeks,decapitating shoots in the hypocotyl, and inserting into hormone-free MSsolid medium supplemented with kanamycin sulfate (100 mg/l). Aftergrowth for two weeks under 5000 lux, resistant plants were clearlydistinguishable from sensitive plants: resistant plants formed roots andgreen leaves, while sensitive plants did not. Results are presented inTable II.

Table II shows actual data and expected data for the self-cross andbackcross (to Wisconsin 38) progeny of eighteen transformants (A-R). Theactual data were obtained as follows. For each cross, seeds (over 100 innumber) were obtained and germinated on moist filter paper. From eachgroup of germinating seeds, 100 plants were selected and decapitated,then inserted into hormone-free MS solid medium supplemented withkanamycin sulfate (100 mg/l). Of the 100 plants for each cross, thenumber of plants which were kanamycin resistant appears in the"KanR/100" column. (For plant B, 80 rather than 100 plants weremeasured; the numbers shown for plant B are adjusted to conform to astandard of 100 plants). The expected data show theoretical values forsingle locus (3:1) and double locus (15:1) insertions of the marker.

As seen from Table II, 14 of the 18 transformants showed segregationdata for both self-cross and backcross progeny consistent with singlelocus insertion. Three of the transformants show data consistent withdouble locus insertion; one transformant (plant A) fits neitherprojection. See the column in Table II captioned "#loci." These resultsshow that it is possible to obtain transformed plants in which theintroduced gene segregates as a single genetic marker locus.

                  TABLE II    ______________________________________    Segregation of Kanamycin Resistance in Nicotiana tabacum    Transformed by Cocultivation with pGV3850kanR    Self-Cross        Backcross    Actual     Expected   Actual    Expected                                            #    Plant         KanR/100  3:1    15:1  KanR/100                                        1:1  3:1  loci    ______________________________________    A    58        75     93.75 38      50   75   ?    B    95        75     93.75 72      50   75   2    C    75        75     93.75 46      50   75   1    D    94        75     93.75 74      50   75   2    E    76        75     93.75 48      50   75   1    F    68        75     93.75 45      50   75   1    G    93        75     93.75 75      50   75   2    H    73        75     93.75 45      50   75   1    I    78        75     93.75 48      50   75   1    J    73        75     93.75 54      50   75   1    K    75        75     93.75 51      50   75   1    L    74        75     93.75 53      50   75   1    M    81        75     93.75 48      50   75   1    N    68        75     93.75 50      50   75   1    O    74        75     93.75 50      50   75   1    P    72        75     93.75 45      50   75   1    Q    76        75     93.75 52      50   75   1    R    72        75     93.75 44      50   75   1    ______________________________________

What is claimed is:
 1. A method of producing a close-linkagedicotyledonous plant containing a marker closely linked to a malesterile locus, comprising:a. transforming a first dicotyledonous plantwith a marker foreign to the species of said first dicotyledonous plantto produce a group of transformants, said marker being substantiallyrandomly inserted into the genome of the plant and said transformingbeing carried out by use of Agrobacterium; b. selecting a group ofsingle locus transformants from the group of transformants; and c.selecting from the group of single locus transformants a close-linkageplant containing a marker in close linkage with a male sterile locus,said close linkage being linkage of less than about 10 recombinationunits.
 2. The method of claim 1 wherein the selecting of a group ofsingle locus transformants comprises selecting by crossing and progenyscoring.
 3. The method of claim 1 wherein the selecting of aclose-linkage plant comprises selecting by crossing and progeny scoring.4. The method of claim 1 wherein the marker is an expressive marker. 5.The method of claim 4 wherein the expressive marker is a visible marker.6. The method of claim 5 wherein the visible marker encodes indigoproduction.
 7. The method of claim 1 wherein the marker is a conditionalexpressive marker.
 8. The method of claim 7 wherein the conditionalexpressive marker is a chromogenic marker.
 9. The method of claim 7wherein the conditional expressive marker is a resistance marker. 10.The method of claim 9 wherein the resistance marker is a kanamycinresistance marker.
 11. The method of claim 7 wherein the conditionalexpressive marker is a dominant conditional lethal marker.
 12. Themethod of claim 11 wherein the dominant conditional lethal marker is amarker encoding indole acetamide hydrolase.
 13. The method of claim 11wherein the dominant conditional lethal marker is a marker encodingrhizobitoxine synthetase.
 14. The method of claim 1 wherein the closelinkage is linkage of less than about 5 recombination units.
 15. Themethod of claim 1 wherein the close linkage is linkage of less thanabout 1 recombination unit.
 16. The method of claim 1 wherein the firstplant is homozygous for male fertility.
 17. The method of claim 1wherein the first plant is homozygous for male sterility.
 18. The methodof claim 1 wherein the first plant is heterozygous for male sterility.19. The method of claim 1 wherein the first plant is selected from thegroup consisting of cotton, rape, cabbage and tomato.
 20. A method ofproducing hybrid seed in a dicotyledonous plant, comprising:a.transforming a first dicotyledonous plant with a marker foreign to thespecies of said first dicotyledonous plant to produce a group oftransformants, said marker being substantially randomly inserted intothe genome of the plant and said transforming being carried out by useof Agrobacterium; b. selecting a group of single locus transformantsfrom the group of transformants; c. selecting from the group of singlelocus transformants a close-linkage plant containing a marker in closelinkage with a male sterile locus, said close linkage being linkage ofless than about 10 recombination units; d. crossing the close-linkageplant and selecting a close-linkage maintainer line heterozygous formale fertility; e. crossing the close-linkage maintainer line with ahomozygous male sterile line to yield a mixture of male fertile and malesterile plants; f. removing male fertile plants from the mixture basedon the presence of the marker to leave a group of plants at least 90% ofwhich are male sterile; and g. pollinating said group of plants at least90% of which are male sterile to yield hybrid seed.
 21. The method ofclaim 20 wherein the selecting of a group of single locus transformantscomprises selecting by crossing and progeny scoring.
 22. The method ofclaim 20 wherein the selecting of a close-linkage plant comprisesselecting by crossing and progeny scoring.
 23. The method of claim 20wherein the marker is an expressive marker.
 24. The method of claim 23wherein the expressive marker is a visible marker.
 25. The method ofclaim 24 wherein the visible marker encodes indigo production.
 26. Themethod of claim 20 wherein the marker is a conditional expressivemarker.
 27. The method of claim 26 wherein the conditional expressivemarker is a chromogenic marker.
 28. The method of claim 26 herein theconditional expressive marker is a resistance marker.
 29. The method ofclaim 28 wherein the resistance marker is a kanamycin resistance marker.30. The method of claim 26 wherein the conditional expressive marker isa dominant conditional lethal marker.
 31. The method of claim 30 whereinthe dominant conditional lethal marker is a marker encoding indoleacetamide hydrolase.
 32. The method of claim 30 wherein the dominantconditional lethal marker is a marker encoding rhizobitoxine synthetase.33. The method of claim 20 wherein the close linkage is less than about5 recombination units.
 34. The method of claim 20 wherein the closelinkage is less than about 1 recombination unit.
 35. The method of claim20 wherein the first plant is homozygous for male fertility.
 36. Themethod of claim 20 wherein the first plant is homozygous for malesterility.
 37. The method of claim 20 wherein the first plant isheterozygous for male sterility.
 38. The method of claim 20 wherein thefirst plant is selected from the group consisting of cotton, rape,cabbage and tomato.
 39. A close-linkage, dicotyledenous maintainer plantheterozygous for male fertility, said maintainer plant comprising adominant marker foreign to the species of said maintainer plant closelylinked in coupling to a male sterile locus at a linkage of less thanabout 10 recombination units.
 40. A method of producing hybrid seedcomprising:a. crossing a line of the close-linkage dicotyledenousmaintainer plants of claim 39 with a line of homozygous male sterileplants to yield a mixture of male fertile and male sterile plants; b.removing male fertile plants from the mixture based on the presence ofthe marker in said male fertile plants to leave a group of plants atleast 90% of which are male sterile; and c. pollinating said group ofplants at least 90% of which are male sterile to yield hybrid seed.